Phytochemical Profile and Antioxidant Properties of Italian Green Tea, A

horticulturae

Article Phytochemical Proﬁle and Antioxidant Properties of Italian Green Tea, a New High Quality Niche Product

Nicole Mélanie Falla, Sonia Demasi , Matteo Caser * and Valentina Scariot

Department of Agricultural, Forest and Food Sciences, University of Torino, Largo Paolo Braccini 2, 10095 Grugliasco, TO, Italy; [email protected] (N.M.F.); [email protected] (S.D.); [email protected] (V.S.) * Correspondence: [email protected]; Tel.: +39-011-670-8935

Abstract: The hot beverage commonly known as tea results from the infusion of dried leaves of the plant Camellia sinensis (L.) O. Kuntze. Ranking second only to water for its consumption worldwide, it has always been appreciated since antiquity for its aroma, taste characteristics, and beneficial effects on human health. There are many different processed tea types, including green tea, a non-fermented tea which, due to oxidation prevention maintains the structure of the bioactive compounds, especially polyphenols; these bioactive compounds show a number of benefits for the human health. The main producers of tea are China and India, followed by Kenya, Sri Lanka, Turkey, and Vietnam, however recently new countries are entering the market, with quality niche productions, among which also Italy. The present research aimed to assess the bioactive compounds (polyphenols) and the antioxidant activity of two green teas (the “Camellia d’Oro” tea—TCO, and the “Compagnia del Lago” tea—TCL) produced in Italy, in the Lake Maggiore district, where nurserymen have recently started to cultivate C. sinensis. In this area the cultivation of acidophilic plants as Citation: Falla, N.M.; Demasi, S.; ornamentals has been known since around 1820. Due to the crisis of the floricultural sector, producers Caser, M.; Scariot, V. Phytochemical have been trying to diversify their product in order to increase their competitiveness, starting to Profile and Antioxidant Properties of cultivate Italian tea. Their antioxidant activity was assessed, finding a similar or higher antioxidant Italian Green Tea, a New High Quality Niche Product. Horticulturae capacity than in other green teas, as reported in literature. TCO showed a higher antioxidant activity 2+ −1 −1 −1 2021, 7, 91. https://doi.org/ (42,758.86 mmol Fe kg ; 532.37 µmol TE g DW; 881.08 µmol TE g DW) and phenolic content −1 2+ −1 −1 10.3390/horticulturae7050091 (14,918.91 mg GAE 100 g DW) than TCL (25,796.61 mmol Fe kg ; 302.35 µmol TE g DW; 623.44 µmol TE g−1 DW; 8540.42 mg GAE 100 g−1 DW). Through HPLC, a total of thirteen phenolic Academic Editors: Lucia Guidi, Luigi compounds were identified quantitatively, including catechins, benzoic acids, cinnamic acids, and De Bellis, Alberto Pardossi and flavonols, in TCO while only 9 in TCL, and mainly in lower amounts. Albeit with differences, both Elazar Fallik teas were found to be of quality proving that Italy could have the possibility to grow profitably C. sinensis. Received: 15 March 2021 Accepted: 23 April 2021 Keywords: tea; Camellia sinensis; antioxidant activity; DPPH; ABTS; FRAP; HPLC Published: 27 April 2021

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional afﬁl- iations. The hot beverage resulting from the infusion of dried leaves of the plant Camellia sinensis (L.) O. Kuntze is commonly known as tea [1,2]. Tea is one of the most popular nonalcoholic beverages consumed across the world, second only to water [3–9], so much appreciated since antiquity for its aroma, taste characteristics, and beneﬁcial health effects [1], thus consumed as an herbal infuse and for its medicinal properties [5,6]. World Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. tea consumption increased to 5.5 million tons by 2016, mostly due to a rapid growth in per This article is an open access article capita income levels in China, India, and other emerging economies [10]. The World Bank distributed under the terms and foresees an increase in average tea auction price from USD 2.80 in 2017 to USD 2.84 in 2020, conditions of the Creative Commons expecting an extension of the global tea market [11]. World tea production reached 5.73 Attribution (CC BY) license (https:// million tons in 2016 [10], with China constituting of 42.6% of world tea production [10], creativecommons.org/licenses/by/ accounting for USD 4 billion [12]. India, the second largest producer, recorded a production 4.0/). of 1.27 million tons in 2016.

Horticulturae 2021, 7, 91. https://doi.org/10.3390/horticulturae7050091 https://www.mdpi.com/journal/horticulturae Horticulturae 2021, 7, 91 2 of 12

There are many different types of teas, and most of them are prepared with the buds of two botanical varieties, C. sinensis var. sinensis and C. sinensis var. assamica (Masters) Wight [9,13]; their characteristics (i.e., appearance, organoleptic taste, chemical contents, and flavor) vary according to the fermentation level: tea can be categorized in nonfermented tea (i.e., green tea, and white tea), semifermented tea (i.e., oolong tea), and fermented tea (i.e., black tea and red tea) [5,6,9,14–17]. The 78% of the tea worldwide production is black tea, especially consumed in Western countries, 20% consists of green tea, which is usually consumed in Asian countries, and 2% regards Oolong tea, commonly consumed in southern China [14]. Globally, the production of green tea increased annually by 5.4% over the past decade, also due to green tea’s perceived health benefits [10]. Actually, fresh tea leaves contain chemical components such as polyphenols (catechins, flavonoids), alkaloids (caffeine, theobromine, etc.), volatile oils, polysaccharides, amino acids, lipids, vitamins (e.g., vitamin C), etc. [6,9,18]. Green teas are produced through steaming or roasting, thus inactivating the activity of polyphenol oxidase, preventing oxidation and so maintaining the structure of the phenolic compounds [1,5,18]. The resulting green teas show a polyphenol content varying from 30% to 42% of dry matter weight [14]. Due to its chemical constituents, tea shows many beneficial properties, such as antioxidant, anti-inflammatory, antiallergic, anticarcinogenic, antidiabetic, and antimicrobial effects [6,7,9,14,15,19–21]. A regular, daily consumption of green tea has been associated with many health benefits [7,22], which are mainly attributed to polyphenols, especially catechins [3,9,23]. Khan and Mukhtar (2007) reported that a balanced diet and the consumption of green tea can protect from oxidative stress and reduce reactive oxygen species damages to lipid membranes, proteins, and nucleic acids. Moreover, many epidemiological studies investigated how tea consumption affected the incidence of cancer in humans, finding a protective and preventive effect of tea against various types of cancer (oral, pharyngeal, and laryngeal cancer) [14], but also healthy effects on many other pathologies involving oxidative stress. Today, more than 50 countries produce different types of tea worldwide, not only as an herbal infuse pleasant to consume, but also for its well-known benefits on human health [2,5]. Tea evergreen plant (C. sinensis)[3,4] is native to South and Southwest China, the Indian Subcontinent, and Southeast Asia [2,24,25], then it became popular in India and Japan, and later in Europe and Russia [5,6]. The first plant specimens arrived in Europe were those studied by Linnaeus in 1763 [26], although the tea beverage had already reached Europe in the XVII century. However, tea cultivation has remained a prerogative of Asian countries, while in Europe the cultivation of the congeneric species Camellia japonica has spread for ornamental purposes only. The first camellia (C. japonica) was introduced in Italy at the end of the XVII century, in Caserta (Campania, South Italy) [27,28]. Since then its cultivation gradually became popular, reaching central and northern Italy, especially Tuscany, Piedmont, and Lombardy [27]. The critical period of the floricultural sector caused by globalization [27,29], since the end of the past decade forced Italian producers of the Lake Maggiore district (Piedmont region) to diversify their final products in order to increase their competitiveness, starting to cultivate C. sinensis in order to produce Italian green tea. Currently there are no studies related to the quality of tea produced in Italy, thus, the aim of the present research was to assess the main bioactive compounds (polyphenols), and beneficial properties (antioxidant activity) of two green teas produced in this new productive context. Horticulturae 2021, 7, x FOR PEER REVIEW 3 of 13

Horticulturae 2021, 7, 91 3 of 12

2. Materials and Methods 2.1. Plant Material2. Materials and Site Characteristics and Methods Camellia sinensis2.1. Plant var. Material sinensis and dried Site leaves Characteristics were kindly provided by “La Compagnia del Lago” and “La CamelliaCamelia sinensisd’Oro” var.plantations,sinensis driedboth located leaves werein the kindly Lake Maggiore provided byarea, “La Compagnia in Piedmont—delNorth Lago” Italy. and Seedlings “La Camelia of camellia d’Oro” derived plantations, from bothacclimatization located in thespecimen Lake Maggiore area, from parks andin botanical Piedmont—North gardens located Italy. Seedlings in the Lake of camelliaMaggiore derived area (i.e. from, Villa acclimatization Taranto, specimen Isola Madre andfrom Villa parks Anelli, and Verbania botanical municipality, gardens located Piedmont in the Lakeregion), Maggiore and were area grown (i.e., Villa Taranto, differently in theIsola two Madre nurseries. and Villa The Anelli,“Compagnia Verbania del municipality, Lago” plantation Piedmont is located region), in the and were grown municipality ofdifferently Premosello in Chiovenda the two nurseries. (VB) (45°55 The′57.6″ “Compagnia N 8°27′16.1″del E), where Lago” the plantation annual is located in average maximumthe municipality temperature of was Premosello 19.8 °C (July Chiovenda showed (VB) the (45highest◦55057.6 monthly00 N 8◦ 27average016.100 E), where the temperature, withannual 30.6 average °C ), the annual maximum average temperature minimum was temperature 19.8 ◦C (July was showed7.8 °C (January the highest monthly showed the lowestaverage monthly temperature, average with temperature, 30.6 ◦C), thewith annual −3.6 °C average), and minimumthe annual temperature average was 7.8 ◦C rainfall was 122.8(January mm (March showed showed the lowest the monthly highest averagemonthly temperature, average rainfall, with −with3.6 ◦279.0C), and the annual mm, while Octoberaverage showed rainfall the was lowest 122.8 mmmonthly (March average showed rainfall, the highest with monthly0.0 mm) averagefor the rainfall, with investigation year279.0 2017 mm, (Figure while October1). The seedlings showed the were lowest planted monthly in the average ground rainfall, at a distance with 0.0 mm) for the of 2.20 m in theinvestigation row, in a stony year 2017sloping (Figure terrain,1). The managed seedlings as werea meadow planted for in more the ground than a at a distance of century. Irrigation2.20 mwas in themade row, by in drip a stony-wing sloping system terrain, when managed occurred. as The a meadow plants were for more not than a century. fertilized. Irrigation was made by drip-wing system when occurred. The plants were not fertilized.

Figure 1. MonthlyFigure 1. average Monthly temperatures average temperatures (maximum (maximum and minimum— and minimum◦C), and— °C monthly), and monthly average average rainfall (mm) for the rainfall (mm) for the investigation year 2017 in: (A). the “Compagnia del Lago” site of cultivation. investigation year 2017 in: (A). the “Compagnia del Lago” site of cultivation. (B). the “Camelia d’Oro” site of cultivation. (B). the “Camelia d’Oro” site of cultivation. The “Camelia d’Oro” plantation is located in Pallanza (VB) (45◦56047.900 N 8◦35012.600 E), The “Cameliawhere d’Oro” the annual plantation average is located maximum in Pallanza temperature (VB) (45°56 was 19.2′47.9″◦C, N the8°35 annual′12.6″ average min- E), where the imum annual temperature average maximum was 9.2 temperature◦C, and the wasannual 19.2 average °C , the rainfall annual was average 135.6 mm for the minimum temperatureinvestigation was year9.2 °C 2017, and (Figure the annual1). Here, average before rainfall planting was the 135.6 seedlings, mm for the the soil was tilled investigation yearwith 2017 a milling (Figure cutter 1). andHere, a bottombefore fertilizationplanting the with seedlings, organic the manure soil was burial tilled powder (Humus with a milling Vita,cutter Fomet and a Spa,bottom San fertilization Pietro di Morubio with organic (VR), Italy)manure (25 burial kg per powder 100 m2 (Hu-. N, P, and K were mus Vita, Fometall presentSpa, San as Pietro 3–4% di of Morubio the total amount,(VR), Italy) accounting (25 kg per on 100 average m2. N, for P, 8.75 and g K of each per m2. were all presentOrganic as 3–4% matter of the accounted total amount, for 38–45%, accounting thus on on average average for 103.75 8.75 g of per each m2 perof it were added.) m2. Organic matterwas made. accounted The plants for 38 were–45%, transferred thus on average to the ground, 103.75 atg per a distance m2 of itof were 0.8 m in a row and added.) was made.1.2 m The in the plants inter-row, were resultingtransferred in 1to plant the ground, per m2. at The a distance hole in the of ground0.8 m in was a made with a row and 1.2 mdrill in the and inter 2.5- Lrow, of Blond resulting peat in were 1 plant added per to m each2. The plant hole as in a soilthe improver.ground was The plants were made with a drilltamped and 2.5 down L of andBlond mulched peat were with added an organic to each mulching plant as a consisting soil improver. in a mix The of wood chips plants were tampedand leaves. down In and May, mulched a cover with fertilization an organic with mulching an organic consisting ox-blood in fertilizer a mix of (10 cc per plant) wood chips and(Biostan, leaves. In Aifar May, Agrochimica a cover fertilization Srl, Ronco with Scrivia an organic (GE), ox Italy)-blood was fertilizer performed. (10 Plants were cc per plant) (Biostan,irrigated Aifar with drip-wingAgrochimica irrigation Srl, Ronco with Scrivia about 1.6(GE), L ofItaly) water was per performed. plant per day. Plants were irrigated with drip-wing irrigation with about 1.6 L of water per plant per day. 2.2. Tea Harvest and Preparation In both plantations, fresh tea shoots with one (late spring or September harvest) or two (spring harvest between late April and early May) tender leaves and a bud were harvested. Horticulturae 2021, 7, 91 4 of 12

After harvesting, tea leaves were steamed for about 1–2 min; once dried, the leaves were roasted and rolled on a hot pan at about 80 ◦C for about 10–15 min to stop the fermentation. They were then left in a dryer at 40 ◦C for 18 h, and ﬁnally stored in plastic containers at room temperature. Thus, green teas were obtained from dried leaves received from: “La Camelia d’Oro” plantation (TCO—Tea Camellia d’Oro), providing us with samples of one shoot with one leaf (ﬁrst harvest), and “La Compagnia del Lago” plantation (TCL—Tea Compagnia del Lago), providing us with a mixture of the two harvests.

2.3. Tea Extract Preparation The dried leaves were ground with a mortar and pestle into a fine powder. Two hundred milliliter of deionized water was heated to 100 ◦C. One gram of dried tea leaves powder was added to the cooling water and left to infuse for 10 min, being stirred ev- ery two minutes. The infusion was filtered with paper filters (Whatman filter papers No. 1, Whatman, Maidstone, UK), and then with polytetrafluoroethylene (PTFE, VWR International, Milano, Italy) filters, with a 25 mm diameter and 0.45 µm pore size. Both infusions were diluted with deionized water to obtain the working solution and maintained at −20 ◦C for the following analysis.

2.4. Bioactive Compounds 2.4.1. Total Polyphenols The total phenolic content of diluted TCL (dilution 1:1 = 1 mL deionized water: 1 mL infusion) and TCO (dilution 1:2 = 1.2 mL deionized water: 600 µL infusion) was determined following the Folin-Ciocalteu method, as indicated by Singleton et al. [30]. The analysis was performed as follows: 1000 µL of diluted 1:10 Folin reagent were mixed with 200 µL of infusion in each plastic tube. The samples were left in the dark at room temperature for 10 min, then 800 µL of Na2CO3 (7.5%) were added to each tube. Samples were left in the dark at room temperature for 30 min. Absorbance was then measured at 765 nm by means of a spectrophotometer (Cary 60 UV-Vis, Agilent Technologies, Santa Clara, CA, USA), and the results were expressed in milligrams of gallic acid equivalents per 100 g of dry weight (mg GAE 100 g−1 DW). Analysis was performed in six replicates per infusion type.

2.4.2. Antioxidant Activity FRAP Assay The ﬁrst procedure adopted to evaluate the antioxidant activity of diluted TCL (1:2 = 1.2 mL deionized water: 600 µL infusion) and TCO (1:3 = 1.5 mL deionized water: 500 µL infusion) was the ferric ion reducing antioxidant power (FRAP) method [31]. The FRAP solution was obtained by mixing a buffer solution at pH 3.6 (C2H3NaO2*3H2O + C2H4O2 in water), 2,4,6-tripyridyltriazine (TPTZ, 10 mM in HCl 40 mM), and FeCl3*6H2O (20 mM). The antioxidant activity was determined mixing 30 µL of diluted infusion with 90 µL of deionized water and 900 µL of FRAP reagent. The samples were then placed at 37 ◦C for 30 min. Absorbance was measured at 595 nm by means of a spectrophotometer (Cary 60 UV-Vis, Agilent Technologies, Santa Clara, CA, USA). The antioxidant activity was plotted against a FeSO4*7H2O calibration curve. Extraction solution (water) without infusion was used as a control sample. Results were expressed as millimoles of ferrous iron equivalents per kilogram (mmol Fe2+ kg−1 DW). Analysis was performed in six replicates per infusion type.

DPPH Assay The second procedure was the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method [32]. The working solution of DPPH radical cation (DPPH˙, 100 µM) was obtained by dissolving 2 mg of DPPH in 50 mL of MeOH. The solution must have an absorbance of Horticulturae 2021, 7, 91 5 of 12

1000 (±0.05) at 515 nm. To prepare the samples, 40 µL of diluted infusion were mixed with 3 mL of DPPH˙. Samples were then left in the dark at room temperature for 30 min. Absorbance was measured at 515 nm by means of a spectrophotometer (Cary 60 UV-Vis, Agilent Technologies, Santa Clara, CA, USA). The DPPH radical-scavenging activity was calculated as: [(Abs0 − Abs1)/Abs0] × 100 where Abs0 is the absorbance of the control (extraction solution without infusion) and Abs1 is the absorbance of the sample. The antioxidant capacity was plotted against a Trolox calibration curve and results were expressed as µmol of Trolox equivalents per gram of dry weight (µmol TE g-1 DW). Analysis was performed in six replicates per infusion type.

ABTS Assay The third procedure was the 2,20-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) method [33]. The working solution of ABTS radical cation (ABTS˙) was obtained by the reaction of 7.0 mM ABTS stock solution with 2.45 mM potassium persulfate (K2S2O8) solution. After incubating for 12–16 h in the dark at room temperature, the working solution was diluted with distilled water to obtain an absorbance of 0.7 (±0.02) at 734 nm. The antioxidant activities of the diluted TCL (1:2) and TCO (1:3) infusions were assessed mixing 30 µL of infusion with 2 mL of diluted ABTS˙. The samples were left in the dark for 10 min. Absorbance was then measured at 734 nm by means of a spectrophotometer (Cary 60 UV- Vis, Agilent Technologies, Santa Clara, CA, USA). The ABTS radical-scavenging activity was calculated as: [(Abs0 − Abs1)/Abs0] × 100 where Abs0 is the absorbance of the control (extraction solution without infusion) and Abs1 is the absorbance of the sample. The antioxidant capacity was plotted against a Trolox calibration curve and results were expressed as µmol of Trolox equivalents per gram of dry weight (µmol TE g-1 DW). Analysis was performed in six replicates per infusion type.

2.4.3. Identification and Quantification of Bioactive Compounds by HPLC The bioactive compounds contained in the infusions were determined by means of two high performance liquid chromatography-diode array detection (HPLC–DAD) method [34], using an Agilent 1200 High-Performance Liquid Chromatograph coupled to an Agilent UV-Vis diode array detector (Agilent Technologies, Santa Clara, CA, USA). Detailed chromatographic methods are reported in Table1. Phytochemical separation was performed with a Kinetex C18 column (4.6 × 150 mm, 5 µm, Phenomenex, Torrance, CA, USA), using different mobile phases for compound identification and recording of UV spectra at different wavelengths, based on HPLC methods, as previously tested and validated [35,36] with some modifications. UV spectra were recorded at 330 nm and 280 nm. The following bioactive compounds were determined: cinnamic acids (caffeic acid, chlorogenic acid, coumaric acid, ferulic acid), flavonols (hyperoside, isoquercitrin, quercetin, quercitrin, rutin), benzoic acids (ellagic acid, gallic acid), catechins (catechin, epicatechin). All single compounds were identified by a comparison and combination of their retention times and UV spectra with those of authentic standards under the same chromatographic conditions. Results were expressed as mg 100 g−1 of dry weight (DW). Horticulturae 2021, 7, 91 6 of 12

Table 1. HPLC methods and relative chromatographic conditions.

Method Classes of Interest Stationary Phase Mobile Phase Wavelength (nm) A: 10 mM KH PO /H PO Cinnamic acids KINETEX-C18 column 2 4 3 4 A pH = 2.8 330 Flavonols (4.6 × 150 mm, 5 µm) B: CH3CN

A: H2O/CH3OH/HCOOH Benzoic acids KINETEX-C18 column (5:95:0.1 v/v/v), pH = 2.5 B 280 Catechins (4.6 × 150 mm, 5 µm) B: CH3OH/HCOOH (100:0.1 v/v) Elution conditions. Method A, gradient analysis: 5% B to 21% B in 17 min + 21% B in 3 min (2 min conditioning time); ﬂow: 1.5 mL min−1; Method B, gradient analysis: 3% B to 85% B in 22 min + 85% B in 1 min (2 min conditioning time); ﬂow: 0.6 mL min−1.

2.5. Statistical Analysis All data were subjected to the statistical analysis for the normality and homoscedastic- ity through Saphiro-Wilk’s test (p > 0.05) and Levene’s test (p < 0.05), respectively. Mean comparisons were computed using the SPSS 25 software (version 25.0; SPSS Inc., Chicago, IL, USA). Correlations among the bioactive compounds of the two teas were calculated by Pearson’s correlation coefﬁcient test by means of PAST 4.03 software. Principal coordinate analysis (PCA)—Biplot was performed using the same software. Eigenvalues were calculated using a covariance matrix among 30 traits as input, and the two-dimensional PCA biplot was constructed.

3. Results The total polyphenol content and the antioxidant activity (FRAP, DPPH, and ABTS assays) of both examined teas are reported in Table2.

Table 2. Total polyphenols, and antioxidant activity of Tea Compagnia del Lago (TCL) and Tea Camellia d’Oro (TCO). Data are presented as mean value ± standard deviation.

Antioxidant Activity Total Polyphenols Tea Type FRAP DPPH ABTS (mg GAE/100 g DW) 2+ (mmol Fe /kg) (µmol TE/g (µmol TE/g Inhibition % Inhibition % DW) DW) TCL 8540.42 ± 105.38 25796.61 ± 951.83 302.35 ± 10.4 46.9 623.44 ± 4.64 94.3 TCO 14918.91 ± 222.31 42758.86 ± 933.85 532.37 ± 5.95 61.2 881.08 ± 1.81 99.8 p ** *** *** *** The statistical relevance is provided (** = p < 0.01; *** = p < 0.001).

The total phenolic content varied from 8540.42 to 14918.91 mg GAE 100 g−1 being signiﬁcantly higher in TCO. The antioxidant activity resulted higher for TCO rather than for TCL (42758.86 and 25796.61 mmol Fe2+ kg−1 for the FRAP assay, respectively; 532.37 and 302.35 µmol TE g−1 DW for the DPPH assay, respectively; 881.08 and 623.44 µmol TE g−1 DW for the ABTS assay, respectively). In the TCO infusion, 13 compounds out of 13 were found by means of HPLC analysis, namely caffeic acid, chlorogenic acid, coumaric acid, ferulic acid, hyperoside, isoquercitrin, quercetin, quercitrin, rutin, ellagic acid, gallic acid, catechin, and epicatechin, while in TCL infusion, only 9 compounds out of 13 were found (chlorogenic acid, coumaric acid, ferulic acid, and quercetin were not detected) (Figure2, Table3). Horticulturae 2021, 7, x FOR PEER REVIEW 7 of 13

Horticulturae 2021, 7, 91 7 of 12 in TCL infusion, only 9 compounds out of 13 were found (chlorogenic acid, coumaric acid, ferulic acid, and quercetin were not detected) (Figure 2, Table 3).

Figure 2. ChromatographicChromatographic profile profile of ofbenzoic benzoic acids acids and and catechins catechins (A), (andA), andcinnamic cinnamic acids acids and flavonols and flavonols (C) in (TCOC) in. Chro- TCO. Chromatographicmatographic profile profile of benzoic of benzoic acids and acids catechins and catechins (B) and ( Bcinnamic) and cinnamic acids and acids flavonols and flavonols (D) in TCL. (D) TC inO TCL. = Tea TCO Camellia = Tea Camelliad’Oro. TC d’Oro.L = Tea TCL Compagnia = Tea Compagnia del Lago. del Lago.

Table 3.3. BioactiveBioactive compounds in Tea CompagniaCompagnia del Lago (TCL) and Tea CamelliaCamellia d’Oro (TCO). Data are presented as mean value ± standard deviation. The values are given in mg 100 g−1 DW. mean value ± standard deviation. The values are given in mg 100 g−1 DW. Flavonols Tea type Flavonols Tea type Hyperoside Isoquercitrin Quercetin Quercitrin Rutin Hyperoside Isoquercitrin Quercetin Quercitrin Rutin TCL 25.37 ± 4.39 35.46 ± 2.60 - 242.38 ± 10.11 44.66 ± 3.93 TCL 25.37 ± 4.39 35.46 ± 2.60 - 242.38 ± 10.11 44.66 ± 3.93 TCO 28.24 ± 4.30 35.31 ± 4.38 388.28 ± 95.47 113.11 ± 14.75 42.32 ± 4.19 pTCO ns 28.24 ± 4.30 35.31ns± 4.38 388.28 ± 95.47 113.11 ***± 14.75 42.32ns± 4.19 p ns nsCinnamic acids *** ns Tea type Cinnamic acids Tea type Caffeic acid Chlorogenic acid Coumaric acid Ferulic acid TCL 42.57Caffeic ± 6.67 acid Chlorogenic- acid Coumaric- acid Ferulic- acid TCOTCL 43.36 42.57± 1.72± 6.67612.25 ± - 37.58 204.62 - ± 16.47 57.85 -± 13.32 p ns TCO 43.36 ± 1.72 612.25 ± 37.58 204.62 ± 16.47 57.85 ± 13.32 Benzoic acids Catechins Tea typep ns Ellagic acid Gallic acid Catechin Epicatechin Benzoic acids Catechins TCLTea type 59.06 ± 2.33 42.39 ± 2.37 122.06 ± 10.86 770.39 ± 21.06 TCO 86.85Ellagic ± 5.17 acid803.88 Gallic ± 56.99 acid 478.98 Catechin ± 27.53 735.84 Epicatechin ± 89.76 p TCL** 59.06 ± 2.33 42.39***± 2.37 122.06 ±***10.86 770.39 ±ns21.06 TCOThe statistical relevance 86.85 ± is5.17 provided (ns 803.88 = non±-significant;56.99 ** 478.98= p < 0.01;± 27.53 *** = p < 0.001). 735.84 -: compound± 89.76 not detected. p ** *** *** ns The two teas showed significant different contents in quercitrin (242.38 mg 100 g−1 The statistical relevance is provided (ns = non-significant; ** = p < 0.01; *** = p < 0.001). -: compound not detected. DW in TCL and 113.11 mg 100 g−1 DW in TCO). TCO and TCL showed significant different contents in cinnamic acids, with TCO being richer in these compounds than TCL. Benzoic acids and catechins were significantly higher in TCO than in TCL (except for epicatechin, The two teas showed significant different contents in quercitrin (242.38 mg 100 g−1 which showed no significant differences−1 in the two infusions). DW inThe TCL phenolic and 113.11 content mg 100in both g DWteas inproved TCO). to TCO be significantly and TCL showed correlated significant to their different anti- oxidantcontents activity in cinnamic (Figure acids, 3), for with the TCO three being methods richer used in these (FRAP, compounds DPPH, ABTS). than TCL.The phenolic Benzoic acids and catechins were significantly higher in TCO than in TCL (except for epicatechin, which showed no significant differences in the two infusions). Horticulturae 2021, 7, 91 8 of 12

Horticulturae 2021, 7, x FOR PEER REVIEW 8 of 13

The phenolic content in both teas proved to be signiﬁcantly correlated to their antioxidant activity (Figure3), for the three methods used (FRAP, DPPH, ABTS). The phenolic content was also positively correlated (p < 0.05) to chlorogenic acid, coumaric acid, ferulic content was also positively correlated (p < 0.05) to chlorogenic acid, coumaric acid, ferulic acid,acid, quercetin, quercetin, ellagic ellagic acid, acid, gallic gallic acid,acid, andand catechin catechin content. content. Interestingly, Interestingly, chlorogenic chlorogenic acidacid was was found found to to be be negatively negatively correlated ( p(p< < 0.05) 0.05) to to the the quercitrin quercitrin content, content, and thisand this latterlatter was was negatively negatively correlated correlated to gallic acidacid too. too.

Figure 3. Pearson correlation among the bioactive compounds of Tea “Compagnia del Lago” (TCL) and Tea “Camelia Figured’Oro” 3. Pearson (TCO). correlation Boxed ellipses among are significantly the bioactive correlated compounds (p < 0.05). of Tea Blue “Compagnia indicates positive del Lago” correlation (TCL) and and red Tea indicates “Camelia d’Oro”negative (TCO). correlation. Boxed ellipses The more are thesignificantly ellipse is compressed, correlated the(p < stronger 0.05). Blue the correlation. indicates 1.positive Polyphenols; correlation 2. FRAP; and 3. DPPH;red indicates 4. negativeABTS; correlation. 5. caffeic acid; The 6.more chlorogenic the ellipse acid; is 7. compressed, coumaric acid; the 8. ferulicstronger acid; the 9. correlation. hyperoside; 10. 1. isoquercitrin;Polyphenols; 11. 2. quercetin;FRAP; 3. 12.DPPH; 4. ABTS;quercitrin; 5. caffeic 13. acid; rutin; 6. 14. chlorogenic ellagic acid; acid; 15. gallic 7. coumaric acid; 16. catechin;acid; 8. ferul 17. epicatechin.ic acid; 9. hyperoside; 10. isoquercitrin; 11. quercetin; 12. quercitrin; 13. rutin; 14. ellagic acid; 15. gallic acid; 16. catechin; 17. epicatechin. The relationship between the studied parameters were evaluated through a PCA andThe represented relationship in abetween two-dimensional the studied PCA parameters scatterplot were (based evaluated on the first through two principal a PCA and representedcomponents in a (PCs)) two-dimensional (Figure4). As PCA depicted, scatterplot the first (based two PCs on explainedthe first two 77.8% principal of total com- ponentsvariation. (PCs)) The (Figure first PC 4). accounted As depicted, for 62.2% the and first was two positively PCs explained correlated 77.8% with of polyphenols, total variation. Theantioxidant first PC accounted activity (FRAP, for 62.2% DPPH, and and was ABTS), positively caffeic correlated acid, chlorogenic with polyphenols, acid, coumaric antiox- acid, ferulic acid, hyperoside, isoquercitrin, quercetin, ellagic acid, gallic acid, and catechin; idant activity (FRAP, DPPH, and ABTS), caffeic acid, chlorogenic acid, coumaric acid, fer- conversely, it was negatively correlated with quercitrin, rutin, and epicatechin. The second ulicPC acid, accounted hyperoside, for 15.6% isoquercitrin, and was positively quercetin, correlated ellagic acid, with chlorogenicgallic acid, acid,and catechin; coumaric conversely,acid, ferulicit was acid,negatively isoquercitrin, correlated quercetin, with quercitrin, quercitrin, rutin, rutin, gallic and acid; epicatechin. conversely, The it was second PC negativelyaccounted correlated for 15.6% with and polyphenols, was positively antioxidant correlated activity with (FRAP, chlorogenic DPPH, andacid, ABTS), coumaric acid,caffeic ferulic acid, acid, hyperoside, isoquercitrin, ellagic acid,quercetin, catechin, quercitrin, and epicatechin. rutin, Thegallic scatterplot acid; conversely, showed that it was negativelythe two teascorrelated are clearly with distinguished, polyphenols, confirming antioxidant that TCOactivity is mainly (FRAP, linked DPPH, to almost and allABTS), caffeicthe detectedacid, hyperoside, bioactive compounds. ellagic acid, In thecatechin, opposite, and TCL epicatechin. was correlated The only scatterplot to quercitrin, showed thatrutin, the two and teas epicatechin. are clearly distinguished, confirming that TCO is mainly linked to almost all the detected bioactive compounds. In the opposite, TCL was correlated only to quercitrin, rutin, and epicatechin. Horticulturae 2021, 7, x FOR PEER REVIEW 9 of 13

Horticulturae 2021, 7, 91 9 of 12

Figure 4. Principal component analysis (PCA)-biplot of Tea Compagnia del Lago (TCL) and Tea Camelia d’Oro (TCO), Figure 4. Principal component analysis (PCA)-biplot of Tea Compagnia del Lago (TCL) and Tea Camelia d’Oro (TCO), by bymeans means of of the the PAST PAST 4.03 4.03 software. software. 1. 1.Polyphenols; Polyphenols; 2. FRAP; 2. FRAP; 3. DPPH; 3. DPPH; 4. ABTS; 4. ABTS; 5. caffeic 5. caffeic acid; acid;6. chlorogenic 6. chlorogenic acid; 7. acid; cou- 7.maric coumaric acid; acid; 8. ferulic 8. ferulic acid; acid; 9. hyperoside 9. hyperoside;; 10. isoquercitrin 10. isoquercitrin;; 11. quercetin 11. quercetin;; 12. quercitrin 12. quercitrin;; 13. rutin 13. rutin;; 14. el 14.lagic ellagic acid; acid; 15. gallic 15. gallicacid; acid; 16. catechin 16. catechin;; 17. epicatechin 17. epicatechin.. 4. Discussion 4. Discussion The Italian tea “Camelia d’Oro” (TCO) showed a total phenolic content (14918.91 mg The Italian tea “Camelia d’Oro” (TCO) showed a total phenolic content (14918.91 mg GAE 100 g−1 DW) higher than the other green teas analyzed in similar studies. Cai et al. [37] GAE 100 g−1 DW) higher than the other green teas analyzed in similar studies. Cai et al. purchased the green tea sample in a Chinese trade market, finding a total phenolic content [37] purchased the green tea sample in a Chinese trade market, finding a total phenolic of 13,600 mg GAE 100 g−1 DW. Mildner-Szkudlarz et al. [38] obtained Yunan green tea content of 13,600 mg GAE 100 g−1 DW. Mildner-Szkudlarz et al. [38] obtained Yunan green leaves in a special tea store, then ground leaves were extracted for 24 h using a 95% ethanol tea leaves in a special tea store, then ground leaves were extracted for 24 h using a 95% solution; the analysis showed a total phenolic content of 13,654 mg GAE 100 g−1 DW. ethanol solution; the analysis showed a total phenolic content of 13,654 mg GAE 100 g−1 TCO even showed a higher phenolic content than the white tea analyzed by Zielinski etDW. al. [39 TCO], which even obtainedshowed a higher wide range: phenolic 3800–11,400 content than mg GAEthe white 100 g tea−1 DW.analyzed Conversely, by Zielinski the −1 “Compagniaet al. [39], which del Lago” obtained tea(TCL) a wide showed range: 380 a total0–11 phenolic,400 mg GAE content 100 of g8540.42 DW. Conversely,± 105.38 mg the GAE“Compagnia 100 g−1 DW, del Lago” lower tea than (TCL) the previous showed a studies’ total phenolic results. content These dataof 8540.42 therefore ± 105.38 might mg −1 indicateGAE 10 that0 g theDW, cultivation lower than and the processingprevious studies’ methods results. adopted These by data “La therefore Camelia might d’Oro” in- plantationdicate that promoted the cultivation the accumulation and processing of phenolicmethods substances.adopted by “La However, Camelia Pé d’Oro”rez-Burillo plan- ettation al. [8] promoted found a higher the accumulation polyphenols contentof phenolic in commercial substances. green However, tea in Pérez Spain,-Burillo brewed et in al. water[8] found for 7 a min higher at 98 polyphenols◦C, obtaining content a value in commercial of 1043 ± 5 green mg GAE tea in L −Spain,1 (TCO brewed = 745.95 in water mg GAEfor 7 L −min1). at 98 °C, obtaining a value of 1043 ± 5 mg GAE L−1 (TCO = 745.95 mg GAE L−1). TheThe antioxidant antioxidant activity activity of of the the two two teas, teas, assessed assessed with with the the DPPH, DPPH, ABTS, ABTS, and and FRAP FRAP methods,methods, confirmed confirmed the the phenolic phenolic trend, trend, being being higher higher in in TCO TCO than than in in TCL. TCL. Regarding Regarding to to thethe DPPH DPPH assay, assay, TCO TCO andand TCLTCL showed values values of of 532.37 532.37 µmolµmol TE TE g−1 g DW−1 DW and and 302.35 302.35 µmol µmolTE g TE−1 DW g−1 respectively,DW respectively, with an with average an average inhibition inhibition percentage percentage of 46.9% of 46.9%and 61.2% and 61.2%respec- respectively.tively. These These resul resultsts are higher are higher than than those those published published by Sirichaiwetchakoon by Sirichaiwetchakoon et al et. [3] al., [who3], whofound found an inhibition an inhibition percentage percentage of 41.46% of 41.46% for commercial for commercial green green tea, purchased tea, purchased in a super- in a supermarketmarket in the in theUnited United Kingdom Kingdom and and prepared prepared by byboiling boiling ground ground tea tea leaves leaves in in 80 80 °C◦C 1x 1xphosphate phosphate buffered buffered saline saline (PBS) (PBS) for for 5 min 5 min.. Conversely, Conversely, Lv Lvet al. et al.[40] [ 40found] found a higher a higher inhi- inhibitionbition percentage percentage in in three three black black teas, teas, ranging ranging from 82.3%82.3% toto 87.6%.87.6%. ThisThis is is unusual, unusual, as as manymany researchers researchers suggest suggest that that green green tea tea has has more more antioxidant antioxidant activity activity than than black black tea tea [38 [38]], , asas green green tea tea has has no no fermentation fermentation processing processing [ 41[41]].. However, However, the the samesame authorsauthors [[40]40] foundfound a alower lower inhibition inhibition percentage percentage through through the the ABTS ABTS assay, assay, ranging ranging from from 12.08 12.08 to to18.08 18.08%,%, ac- accordingcording to to the black teatea type.type. Conversely,Conversely, inin thisthis study study the the ABTS ABTS assay assay confirmed confirmed the the DPPHDPPH high high values values for for both both tea tea samples, samples, ranging ranging from from 94.3% 94.3% in in TCL TCL to to 99.8% 99.8% in in TCO, TCO, thus thus confirmingconfirming the the highest highest antioxidant antioxidant activity activity of of green green tea. tea. Horticulturae 2021, 7, 91 10 of 12

A few works also evaluated the antioxidant activity by means of FRAP test, however, using different units of measurement, making it complex to compare the different values [8,9]. The “Camelia d’Oro” tea (TCO) resulted to contain all the 13 bioactive compounds investigated, while the “Compagnia del Lago” tea (TCL) showed only 9. The major components identified in both Italian teas were quercetin, catechin, and epicatechin; in TCO also chlorogenic acid and gallic acid. Although not giving the single related values, Cai et al. [37] also found that catechin, epicatechin, and quercetin were the major types of phenolic compounds found in tea leaves. The “Camelia d’Oro” tea (TCO) showed catechins (478.98 mg 100 g−1 DW) and epicatechins (735.84 mg 100 g−1 DW) values (Table3) similar or higher than other green teas. Li et al. [9] found in the green Longjing tea values of 0.36 mg 100 mg−1 DW for catechins and 0.90 mg 100 mg−1 DW for epicatechins. Hyun et al. [25] found lower catechin values (ranging from 3.14 to 4.76 mg g−1 FW), but higher epicatechin values (ranging from 8.12 to 10.40 mg g−1 FW) than TCO. Conversely, the “Compagnia del Lago” tea (TCL) showed catechins (122.06 mg 100 g−1 DW) and epicatechins (770.39 mg 100 g−1 DW) values lower than the previous studies results. It is noteworthy that the catechin content is highly correlated to the antioxidant activity measured with the three assays (FRAP, DPPH, and ABTS) (Table3), as already found by Pérez-Burillo et al. [8], when comparing white and green teas. Catechins are known to have important human health benefits, due to their antioxidative, anti-inflammatory, anticarcinogenic, antidiabetic, and antimicrobial properties [34,41], and they may also help reduce the body mass index [42]. Quercetin shows antihypertensive effects, improving endothelial function, gene expression, and modulating cell signals [43], while coumaric acid has protective effects against carcinogenesis, atherosclerosis, oxidative cardiac damages and has anti-inflammatory effects [44]. Gallic acid of TCO (803.88 mg 100 g−1 DW) was higher than the Longjing tea values (0.07 mg 100 mg−1 DW) [9], conversely TCL (42.39 mg 100 g−1 DW) values were lower. Although not showing the single related values, Gorjanovićet al. [45] showed the presence of ferulic and caffeic acids in green tea infusions, as they were detected in this study (Table3). Flavonols (hyperoside, isoquercitrin, quercitrin, rutin), chlorogenic acid, and ellagic acid were not frequently reported individually in other studies, but more as a single category of flavonols, cinnamic acids, and benzoic acids; they were almost all found in both the Italian teas. Flavonols are health-promoted compounds [42] which can be largely found in green tea leaves [46], and chlorogenic acid has antioxidant, anti-inflammatory, anticancer, an- tilipidemic, antidiabetic, antihypertensive, and antineurodegenerative activities [47], thus these teas are a rich source of bioactive compounds. As already observed by Li et al., Lv et al., and Tenore et al. [9,21,40], antioxidant activity was positively correlated with polyphenol content and, thus, with catechins content. The methods for the antioxidant activity analysis were also positively correlated with each other, as Zielinski et al. [39] observed.

5. Conclusions This work investigated the content in bioactive compounds of two Italian teas, both cultivated in the Lake Maggiore District, in Piedmont. The results indicated that the two teas have different phenolic compositions, probably due to the different cultivation substrates or techniques. The “Camelia d’Oro” tea (TCO) showed in general, higher bioactive compounds levels than the “Compagnia del Lago” tea (TCL). However, both teas showed values in accordance with other studies’ results, or even higher, conﬁrming that they would be suitable to diversify the Italian growers’ production with the C. sinensis cultivation. Horticulturae 2021, 7, 91 11 of 12

Author Contributions: Conceptualization, V.S.; methodology, M.C.; investigation, N.M.F., S.D., M.C.; data curation, N.M.F., M.C.; writing—original draft preparation, N.M.F.; writing—review and editing, M.C., S.D., V.S.; supervision, V.S.; project administration, V.S.; funding acquisition, V.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: Authors acknowledge Dario Donno for HPLC analysis, and the two nurseries, “La Camellia d’Oro” and “La Compagnia del Lago” for providing the two tea leaves. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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